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Chromosomal rearrangements of the 1 lpl5 locus have been identified in hematopoietic
malignancies, resulting in translocations involving the N-terminal portion of the nucleoporin
gene NUP98. Sixteen different fusion partner genes have been identified for NUP98 and over
half of these are homeobox transcription factors. By contrast, the NUP98 fusion partner in
t(l 1;20) is Topoisomerase I (TOPI), a catalytic enzyme recognized for its key role in relaxing
supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in
murine bone marrow (BM) confers a potent in vitro growth advantage and a block in
differentiation in hematopoietic precursors. In a murine BM transplantation model, NUP98-
TOP1 expression led to a lethal, transplantable acute myeloid leukemia (AML). To ascertain if
NUP98-TOP1 acts through a novel pathway, a panel of NUP98-TOP1 mutants was engineered
and tested for their sub-cellular localization and their growth promoting effects. Neither the
NUP98- 5' nor TOP1-3' portion of the fusion alone, nor a novel VP16-TOP1 fusion had any
growth enhancing effects. Moreover, mutants lacking TOPI domains known to be involved in
DNA binding were also unable to transform myeloid progenitors. The TOP1-3' mutant
exhibited ubiquitous GFP expression, while NUP98-5' and the DNA binding mutants localized
to distinct nuclear bodies. In-vitro mutagenesis was employed to mutate the TOPI active-site
tyrosine (Y723F), a mutation known to abolish TOPI catalytic activity. Similar to NUP98-
TOP1, NT-Y723F exhibited a nuclear localization, had an in vitro growth advantage and
induced a lethal, transplantable AML, suggesting that NUP98-TOP1 induces its leukemogenic
effects independent of TOPI catalytic, isomerase activity.
As observed with expression of other translocation fusion products, the long latency of
disease onset suggests the acquisition of additional genetic mutations. Two approaches were
used to identify potential NUP98-TOP1 collaborating genes. We chose the strong candidate
gene Meisl, as it has previously been shown to accelerate leukemia induced by several NUP98-
HOX fusions. However, no evidence for collaboration between Meisl and NUP98-TOP1 was
observed. Our second approach followed the serendipitous finding of NUP98-TOP1 retroviral
integration into the ISCBP locus in a leukemic mouse. Strikingly, NUP98-TOP1 expression in
ICSBP deficient bone marrow accelerated disease onset.
The results of this thesis add to the recognition of NUP98 fusion genes as an important
class of leukemic fusion proteins. These studies further demonstrate the complexity of the
molecular pathways involved in leukemogenesis.